Characterization of c-lil, a chicken cellular sequence associated with a stock of B77 avian sarcoma virus.

0022-538X/82/090925-10$02.00/0

CopyrightC 1982,AmericanSocietyfor Microbiology

Vol.43,No. 3

Characterization

of

c-lil,

a

Chicken Cellular Sequence

Associated with a Stock of B77 Avian Sarcoma Virus

M.BOCCARA,t N. PLUQUET, J. COLL, C. ROMMENS, AND D. STEHELIN* Laboratoire d'Oncologie Moleculaire, InstitutPasteur, 59Lille, France

Received 22 March 1982/Accepted 27 May 1982

Using biochemical methods, we have shown that a new specific sequence,

v-lil,

is associated with a given stock of B77 avian sarcoma virus (clone 9). We prepared

aDNAcomplementary tov-lilsequences, using substractive hybridizations, and

investigated the properties of this sequence. v-lilhas a genetic complexity of ca. 2,000 nucleotides and is not present in various stocks of avian sarcoma virus,

avian leukosis virus, or defective leukemia virus.v-lilis not associated with B77

avian sarcoma virus isolated from the original tumor and thus has been acquired

byinvitropassage of the virus on chicken embryo fibroblasts. A search for the

origin ofthev-lil sequence among the DNAs of different avian species has shown

that asimilarsequence,c-lil,is present in normal chicken DNA (1 to 2 copies per

haploid genome). c-lil is not highly conserved but is present in the DNA of all

chickens fromthe genus Gallus. Thec-lil sequence is transcribed at a low level (1 to3copiesper cell) in normal chicken embryo fibroblasts. The biological function, if any, of v-lil or its cellular equivalent has yet to be determined.

The genome of avian leukosis viruses¢ALVs)

hasbeen shown to contain three genes

implicat-edin the replicative functions of the virus: gag

(internal group-specific antigen); pol (reverse transcriptase); and env (envelope glycoprotein) (4). The avian sarcoma viruses (ASVs) possess

either an additional gene, src (Rous viruses

[RSVs]),

or distinct specific sequences (onc

genes) involved in the fibroblastic

transforma-tion ofhost cells (8, 16, 18, 20). Similarly, the

defective leukemia viruses (DLVs) contain

dif-ferent onc genes,accordingtotheir

pathogenici-ty (5), insertedin a deleted ALV genome (13).

Hybridization techniques have allowed stud-iesoftheoriginand evolution ofviralgenesand

their homologous cellular counterparts. It has been shownthat sequences relatedtothe

repli-cative genes of ALV are present in normal

chickencellular DNA(3, 4,8, 12).These

endog-enous viral (ev) sequences, which have been

called evi to evl3 (1), have been

acquired

re-cently by chicken

cells;

indeed,

these sequences

were shown tobeabsentinexotic chickens(4),

andAstrinetal. havebreda roosterwhich lacks

endogenous ALV (1). In contrast, the cellular

homologs of the src sequence from RSV and

onc sequences from DLV have been shown to

be highly conserved

throughout

the

phylogeny

ofhighervertebrates(12,

15,

17, 20).

Until now, the only recombinant viruses

de-tPresentaddress:Departmentof ViralOncology,

Memori-alSloan-KetteringCancerCenter,NewYork,NY 10021.

scribed in the chicken were obtained by

recom-bination between exogenous viral genes and

their cellular endogenous counterparts (7,

23-25) orbetween ALV-related viral sequences and

phylogenetically stable cellular genes, which generated a highly oncogenic virus (6, 12, 13, 15, 16, 18, 22). We present here the finding that

stableassociation between aretrovirusgenome

and acellular sequence which does not appear to

behighly conserved inevolution can occur.

This sequence,calledv-lil, has been foundby

using biochemical methods(20).Weprepareda

src probe from the stock ofB77ASV clone 9.

Afterselection, we analyzedthe characteristics

ofthis cDNAsrc. We found that this probe hy-bridizedtoPragueC strain(PrC)-RSVRNAtoa

plateau value of50% and to aplateau value of

90% with the template B77 RNA. This was

unexpectedsince both strains have been

report-ed to be of similar genetic structure. A likely

explanation was that the RNA from acellular

sequence associated with the RNA from our

stock of B77 ASVwas reverse transcribed and

copurified with the cDNAsrc.

According

tothe

procedure we used for

selecting

the cDNAsrc

andtothekinetics we

obtained,

this shouldnot

beaheterogeneous

population

of cellularRNAs.

Therefore, wedecidedto

purify

thecDNA

spe-cific for this sequence. Here we describe the

purification of this probe, called cDNAtil, and themeasurementof its

complexity.

The

hybrid-ization of

cDNAj,,

withdifferent cellular DNAs

elucidated the

origin

of this sequence.

925

on November 10, 2019 by guest

http://jvi.asm.org/

MATERIALS ANDMETHODS

Cells and viruses. ViruseswerepropagatedonC/E chicken fibroblasts (Brown Leghorn, Institute

Gus-taveRoussy, Villejuif, France).Ahigh-titerisolateof Bratislava 77 ASV (subgroup C) was originally ob-tained from P. Vogt and has been cultured after cellularrecloning (B77clone9)forseveralyears inthe laboratoryof M. Bishop (SanFrancisco, Calif.). After repeatedpassage,thisvirus consistedmainlyof trans-formation-defective (td) variants (90%o of particles; unpublished data) but retained ahigh titer of trans-forming virus (107 focus-forming units perml). This virus stockwas used for thepreparation ofcDNA,j, and willbe called B77 ASV(lit).

Weprepared another B77 ASVfromanearly pas-sage (gift of J. Huppert, Villejuif, France) of the original tumor. Other B77 ASV cultures have been providedbyH. Temin(Madison, Wis.),C.Moscovici (Gainesville, Fla.),R.Friis(Giessen,WestGermany), L. H. Wang (New York, N.Y.), and J. A. Wyke (London, England).

The other viruses were obtained as follows: the Prague B (PrB) strain of RSV from R. Junghans (Pasadena, Calif.); the Schmidt-Ruppin strain,

sub-groupD, ofRSV andreplication-defective(rd) Bryan RSV from H. Rubin(Berkeley, Calif.); PrCRSVfrom University Laboratories (Bethesda, Md.); the Carr-Zilber strain of RSV, Rous-associated virus (RAV)-0 (subgroup E), RAV-2 (B), RAV-49 (C), RAV-50(D), td B77 ASV, and td PrC RSV from P. Vogt (Los Angeles, Calif.) through M. Bishop (San Francisco, Calif.).

The following DLVs were provided by T. Graf (Heidelberg, West Germany):the avian erythroblasto-sisvirus, the fourmyelocytomatosis viruses (MC29, OKIO,MH2,andCM2),andtheavianmyeloblastosis viruses (AMV and E26).

Preparation of viral RNA.Supernatant from infected cellswasharvested,andafterclarification (9,000 x g

for10 minat40C), virions werepelleted(19,000rpm

for90minat4°CinaSpinco L19 rotor) and suspended

inSTE buffer (0.1 M NaCl, 0.001 M EDTA, 0.02 M

Tris-hydrochloride, pH 7.4)with 200 ,ug of proteinase K(Boehringer Mannheim Corp.)perml and1% sodi-umdodecyl sulfate. After digestion for10minat370C, theviralRNAwasextracted twice withSTE-saturated phenol,andtheaqueousphasewasprecipitated

over-night at -20°C with 2 volumes of ethanol in the

presenceof0.2Msodiumacetateand 50,ugofyeast RNApermlascarrier.

For quantitative analysis, the viral RNA was

dis-solved in hybridization buffer (0.6 M NaCl, 0.02 M Tris-hydrochloride [pH 7.4], 500 ,ug ofcalfthymus DNAperml).

Toprepare70SRNA, the viralpelletwasdissolved

in STE. The 70S RNA was separated from

low-molecular-weight RNAs by sedimentation through a

15 to30%o sucrose-STEgradientinanSW41rotor at

40,000rpmfor3.5 hat40C. The70S RNA peakwas

detected byUV spectralanalysis(ISCO,modelUA5), pooled, and ethanol precipitated.

Preparationof cellular DNAs. CellularDNAs from

various avianspecieswereobtainedfrom11-to15-day embryos, tissue culture cells, or erythrocytes. The

detailed procedure has been describedbyFrisbyetal.

(4).

Preparation of cellular RNA. Tissue culture cells

(>50% confluent 100-mm dishes) were suspended in

STEcontaining200,ugofproteinaseKpermland 1%

sodiumdodecylsulfate. Afterincubation for 10minat

37°C,theRNAwasextractedtwice with

STE-saturat-ed phenol and ethanol precipitated. The DNA was

spooled outand discarded. After centrifugation, the RNA pellet was dissolved in STE to a final RNA concentration of 10mg/ml.Theabsorbance at 260 nm/ absorbanceat280nmratiowasfound to be:1.95, and DNAcontamination was measured by the diphenyl-amine testto be less than 10%. The integrityof the RNAwasshownbycentrifugation througha15 to30% STE-sucrosegradientunder conditions which detect-ed therRNAprofile.

Preparation ofvirus-specific single-strandedcDNAs.

[3H]cDNA,il and [32P]cDNA,,p were synthesized by

using purifiedAMVreversetranscriptaseinareaction

containing70SB77(lil)RNAas atemplatetomakea

cDNAj,,,ortdPrB RNAtomakeacDNArep (comple-mentary to all replicative genes of td PrB RNA).

Oligomers of calfthymusDNAwereusedasprimers

(21).Thecompletereactionmixturewasessentiallyas

describedbyShanketal.(14):50 mMTris(pH 8.1);2

mMdithiothreitol; 3mM MgCl2;50 mM KCI;100 ,ug

ofactinomycinDperml;0.1 mM eachdGTP, dATP,

anddCTP; 2mg of calfthymusDNA primerperml;

280 U of AMV polymerase per ml (J. Beard, Life Science Inc.); and 20to40jigof70S templateRNA per ml inafinal volume of 500 ±lfor[3H]cDNAand 125 ,ul for [32P]cDNA. We used 3 mCi of[3H]dTTP

(0.8 mM, 60Ci/mmol, ICN Pharmaceuticals) and 0.5

to 1 mCi of [32P]dTTP (6 ,uM, 610 Ci/mmol, New

EnglandNuclearCorp.).After 90 min ofincubationat

41°C, the reaction was stopped by the addition of

sodiumdodecyl sulfate (1%finalconcentration). The mixturewasincubated for 1 hat37°C with 100 ,ugof proteinase K per ml, extracted with STE-saturated

phenol inthe presence of 200,ugofyeastRNA,and

ethanol precipitated. Thespecific activitieswere 2 x

10' cpm/,ug for [3H]cDNA and 108 cpm/p.g for

[32P]cDNA. These specific cDNAs were then

sedi-mented througha5 to20%alkaline sucrosegradient

(0.9M NaOH, 1 M NaCl,0.01 M EDTA)for 24 hat

110,000xgat240C. The cDNAs ranged in size from4

to 8S, and a size poolof 5 to 7S was used for the selection ofspecific probes. The detailed procedure for[3H]cDNAsrc preparationhas been described

previ-ously(19).

Nucleicacidhybridization.Thestandard solution for hybridization contained 0.6 M NaCl, 0.02 M Tris-hydrochloride(pH 7.4), 0.01 M EDTA, 500 p.g of calf

thymusDNA perml,2,000 cpm (0.04 ng) of[3H]cDNA

or2,000cpm(0.005 ng)of[32P]cDNA,and the appro-priate RNA or DNA in at least 10- to 1,000-fold excess. Incubationswere carried out at 68°C in glass capillaries (Brandt). The extent of annealing was de-terminedafterS1 nuclease digestion (9).

Chromatography onHAP. Chromatography of nu-cleic acids on hydroxylapatite (HAP) columns was carriedout aspreviouslydescribed (20), using 1 mg of packedHAP(Bio-GelHTP, Bio-RadLaboratories) for 200,ugofnucleicacid. For selection probes, two types ofprocedureswereused.

(i) Negative selection (recovery of single-stranded cDNA). To stabilize the hybrids, the column was maintained in a water bath at 50°C. Samples were

on November 10, 2019 by guest

http://jvi.asm.org/

c-lil, A CHICKEN CELLULAR SEQUENCE 927

loadedin 10 mMphosphate buffer (pH 6.8) containing 1.5 M NaCl. Single-stranded cDNAs were eluted by usingalineargradient of phosphate buffer, pH 6.8(0.1 to0.2 M in1.5 MNaCI).The temperature was shifted to 60'C, and the column was washed with 0.15 M phosphate buffer to elute partial hybrids. Double-stranded nucleic acids were recovered with 0.4 M phosphate buffer.

(li) Positive selection (recovery of cDNA hybridized with homologous RNA). Chromatography was con-ducted under conditions which eliminated partial hy-brids before the elution of double-stranded nucleic acids. The column was maintained at 60°C, and sam-pleswereloaded in 10 mM phosphate buffer without NaCl. Single-stranded cDNA and partial hybrids were eluted by 0.14 M phosphate buffer. In both proce-dures, we measured theradioactivity of a sample of eachfraction, tested it forS1nucleaseresistance, and then determined residual hybridization of the recov-ered single-stranded cDNA with the selective RNA.

Determination of geneticcomplexity.Thecomplexity ofcDNA,,,wasdetermined by a method described by Young et al. (26) and adapted by Saule et al. (13), based on the fact that the complexity of a given radioactive cDNA can be defined experimentally by

the Cotia ofthe hybridization kinetics between this

cDNAin excess and thehomologous nonradioactive RNA. To meet this condition, the concentration of B77(li)RNA waspreciselydetermined by a classical Crt curve with

[32P]cDNA,P.

The concentration of RNA must belower than the concentration of

cDNA,j,

tobe inDNA-driving condition, but greater than the concentration of

[32P]cDNA,,p

tobeinCrtcondition. Hybridizationswereconducted at68°Cin 0.6 MNaCl from 150 s to 32 h with 0.2 ng of[3HjcDNA,,,(10,000 cpm), 0.005 ng of

[32P]cDNA,,p

(2,000 cpm),and the required amounts of viral RNA in a final volume of 1 ,ul perpoint.

Determination of thermal denaturation. The prepara-tion of3H-labeled unique-sequencechicken DNA has been described by Frisby et al. (4). The DNA of normalchickencells washybridizedwith[3H]cDNA,wj

or with 3H-labeled unique-sequence chicken DNA (2,000 cpm per point) to a Cot value of 20,000

mol-sliter-1 at afinal DNA concentration of 0.3 M NaCl. A total of 20,000 cpm of [32P]cDNAr,p was

annealed to normal chicken DNA and added as a control, and thesamplewasdivided into 10aliquots, each inasealedcapillary. Thecapillarieswere incu-bated in a water bath equilibrated to the required temperature for 10min,and the percentage of cDNA

step step

70S RNA I1 sscDNA 2

B77 (lil) AMVpolymerase HA

[3H]dTTP PrC RNA hybrids

tdB77 RNA

remaining annealed to the cellular DNA at each de-fined temperature was determined by S1 nuclease digestion (9).

RESULTS

Preparation of cDNAw. DNAspecific forv-lil

was prepared by transcribing the70S RNAfrom

our stock of B77 ASV (lit) grown on chicken

embryo fibroblasts (CEFs). We then selected

[3H]cDNAwhich couldnothybridizetothe70S

RNA from PrC RSV and td B77-C ASV. The

viralstrains used for the selection werecongenic

and of the same subgroup, to eliminate possible

contamination of cDNA15, by cDNA from viral

mutatedsequences. The strategyofpreparation

ofcDNA111 was based on subtraction

hybridiza-tion (Fig. 1). After synthesis, the cDNA was

sedimented through an alkalinesucrosegradient

toeliminate cDNA smaller than 5S or largerthan

7S which would not be appropriate for correct hybridization.

(i)Negative selection. In this step, we selected

thecDNA which was notcomplementary to PrC

and tdB77-C RNAs. Single-strandedcDNA (2

,ug) was hybridized to an excess of PrC (10 ,ug)

and td B77-C RNAs (5 ,ug) under nonstringent

conditions (1.5 M NaCl at 60°C) to reach aC.tof

10

mol'sliter-1

and a

Cot

of 2

mol's

liter-1

and

thenwasseparated from hybrids by

chromatog-raphy on HAP. The single-stranded cDNA was

elutedby a lineargradient of sodium phosphate

buffer (0.1 to 0.2 M) containing 1.5 MNaCl. It

was 2% resistant to hydrolysis by S1 nuclease

and did nothybridize with PrC or td B77RNAs

(<5%S1 resistance at a Crtof20mol

s-liter-1),

but it did hybridize to B77

(lit)

RNA with a

plateau of 50% hybridization, indicating the

presence of DNAcomplementary to sequences

other thanv-lilin thesingle-strandedDNA,orof

sequences too damaged to hybridize to this

RNA.

(ii) Positive selection. In this step,

single-stranded cDNA was

hybridized

to 70S RNA

from B77 ASV

(lit)

at alow Crt (2mol-s

liter-1)

understringentconditions(0.6MNaClat

68°C)

toavoid thehybridizationofnonspecificcDNA

step

ss cDNA 3 sscDNA (cellular contaminants)

HSAP

stepds

__|________t1

or

Sihybrids

4 ,

cDNAjj1i

I

B77(II)RNA

OH-FIG. 1. PreparationofcDNAj,,. Single-strandedcDNA was prepared by reverse transcription of 70S RNA

fromnondefectiveB77ASV(lit)andcentrifugationthroughanalkaline sucrose gradient to select 5 to 7S cDNA (step1).Instep2,single-strandedcDNAwasannealed toanexcessof PrC and td B77 RNAs and separated from hybrids by HAP chromatography. In step 3, this single-stranded cDNA, annealed to the template RNA to eliminatecellularcontaminants,wasrecoveredashybridsafterasecond HAP chromatography (or

S1

digestion).

cDNA,,,wasobtainedafteralkali treatment of thehybrids (step 4).

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.501.58.457.553.619.2]

928 BOCCARA ET AL.

withcontaminatingcellularRNA presentin the

viral RNApreparation. The

cDNA,jj

was eluted

asdouble-strandhybridonHAP and after

dena-turation wasfully sensitivetoSi nuclease

diges-tion. At this step, the cDNA,,1 obtained after

hybridizationwith B77(lil)RNAunderstringent

conditions (0.6 M NaClat68°C) followedbySi

nuclease treatmentinstead of elutionon aHAP

column had the same properties. Recoveries

were similar to those described previously for

cDNAsrc (19), and

cDNAj,,

represented 7% of

theinitial total probe.

The specificity of cDNA,,1 was tested by

hy-bridization with PrC RNA, td B77 RNA, and B77 (lil) RNA (Fig. 2).

[32P]cDNArep

and

[3H]cDNAs,7

were included in the reactions as

internal standards for hybridization. Only the

parental RNAhybridizedwith

cDNAji,,

whereas

all three RNAs fully hybridized with cDNArep.

As expected, B77 (lit) RNA and PrC RNA

hybridized to cDNAsrc.

GeneticcomplexityofcDNAw.

According

toa

modified technique of Youngetal. (13,26), the

complexity of [3H]cDNA111 can be defined by

using the

Cotj12

drawn from the hybridization

kinetics between this cDNA in excess and the

homologous nonradioactive RNA. This method

presented two advantages over the method

which used

32P-labeled

RNA (19): it was not

necessary touse a purified RNA specific to the

cDNA nor to know the ratio between helper

virusRNAand specific RNA.

Wedeterminedprecisely theconcentrationof

specific RNA and chose RNA concentrations corresponding to 20 and 40% hybridization with

[3H]cDNAwjj.

Inadditionto

[3H]cDNA,j1

and B77

(lil) RNA, each sample contained

[32P]cDNArep

as an internal control which would allow us to

construct a Ct curve and to make a precise

determination of the RNA concentration in the

hybridization reaction. If the RNA were in

excess over the two probes, the Crtl/2 and the

Cotj12

would beidentical,and theapparent

com-plexity would be smaller than expected. If the

[3H]cDNA,11 were in excess, the

Cot1/2

shouldbe

constant, and the hybridization plateau value

should be a function of the amount of RNA in

the reaction mixture (Fig. 3A and B). We found

a

COtj12

value of 4.5 x

10-3

mols4liter-1. Since the

Crt1/2

of PrB RNAwas2 x 10-2mol-sliter-1

and corresponded to a complexity of 10,000

nucleotides, we determined that

cDNA1j,

had a

complexityof 2,250 nucleotides ± 10%.

Specificity

of cDNAw. We searched for the

presence of thelid sequence in RNA from ALV,

DLV, and strains of RSV otherthan the ones

used to prepare

cDNA1j,. [3H]cDNA1j,

was

hy-bridizedat aCrt of 5 molsliter-lunder stringent

conditions to different viral RNAs (Table 1). It is striking to see that cDNA,,1 hybridized only with

the B77 RNAused to prepare the probe.

Irre-spective of the group (RSV, ALV, DLV) orthe

subgroup (B, C, D, E) tested, the percentage of S1 resistance was no more than 8%. We

elimi-nated the possibility that these RNAs were

degraded, since

[32P]cDNArep

used in the same

experiments extensively hybridized with all

test-edRNAs.

v-illsequence becomes associated with B77ASV

afterrepeatedpassage inCEFs.v-lil was present

FIG. 2. Hybridization of viral RNAs with

cDNA,j,.

Inparallel experiments,

[3H]cDNAj,,

and

[32P]cDNA,,,

or

[3H]cDNAsrcand

[3cP]cDNArP

were hybridized understringent conditions (0.6 MNaCI, 68°C) todilutions of various RNAs in a volume of 7,ulfor 16 h. The extent of annealing was measured by Si nucleasedigestion.

Symbols: 0,

[3H]cDNA,,,; U, [3H]cDNAsrc;

+, [32P]cDNArep. Crt

(Mxsx1-l)

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.501.109.395.452.634.2]

c-lil, A CHICKEN CELLULAR SEQUENCE 929

50j_ I

/#

/I

/

'I

/

...,

...a A a !..-2

4_-. L-A . Cot

10U' iloU 1o-, (Mxs x1J')

'"'I"II' 't11CCe

lo-, 2 10- 10o

FIG. 3. Geneticcomplexity ofcDNA,11. ToconstructtheCotcurve,cDNA,11 inexcess washybridizedtoB77

(lil) RNA. TheCrt curvewas determined with the concentration ofthe RNA in excesswith regard to the

concentration of the

[32P]cDNAep.

Hybridizationwasconductedat68°Cin0.6 MNaCl from150sto32 h with

0.2ngof

[3H]cDNAj,i

(10,000cpm), 0.005ngof

[32P]cDNA,,p

(2,000cpm),and0.1ng(A)or0.05ng(B)ofviral

RNA ina1-il finalvolumeperpoint. Theextentofhybridizationwasmeasured by resistancetoS1 nuclease.

[image:5.501.51.453.61.249.2]

Symbols: 0,[3HJcDNA,il; +, [32P]cDNArp.

TABLE 1. Hybridizationsof viral 70S RNA to

cDNA,j/'

b%Hybridizationto:

Virus RNA(subgroup) CDNA

cDNA,il

ALV RAV-0 (E) 74 5

PrBtd 95 4

RAV-2(B) 92 7

RAV49(C) 89 6

RAV-50(D) 91 9

B77td(C) 100 4

ASV SR D 74 8

nd B77(C) 95 100

nd PrB 100 4

CZ 91 3

rd BRY 77 8

DLV AEV(RAV-2) 84 6

MC29(RAV-2) 76 7

MH2(RAV-3) 70 12

OK10(RAV-2) 78 7

CM2 (CMAV) 75 9

AMV 61 3

E26(RAV-2) 59 6

a

Hybridization

reactions were performed under

stringentconditionsat afinalCrtof 5 mol-sliter'.The values werenormalizedtothefinal extentof

hybrid-ization withanhomologous RNA: 85% forcDNA,,p

with tdPrB RNA and80%oforcDNA,,,with B77(1il) RNA. The valueof 12%hybridizationwith MH2 RNA hasbeen shownbykineticanalysisof the reactionto beinsignificant.

bSRD, Schmid-Ruppin RSV,groupD;nd,

nonde-fective; CZ, Carr-Zilber RSV; BRY, Bryan RSV; AEV,avianerythroblastosisvirus.

inB77 ASVbutwasnotfoundtobeassociated

with any other tested viruses. To determine

whetherv-lilwaspresentinthe B77 ASV

isolat-ed from the original tumor or if it became

associated with B77 ASV after repeated

pas-sages in tissue culture, we prepared B77 ASV

from an early passage of the original tumor.

Tumor extracts wereinoculated into 1-day-old

chickens(C/E), and after 3 weeks,the

second-ary tumor was removed. CEFs were infected

with a filtrate from this tumor, and the viral

RNA which was produced was analyzed by

hybridization with

[3H]cDNA,jj

and

[32P]cDNArep.

cDNA,,, failed to hybridize with

the viralRNA isolated from the original tumor

(Fig. 4). This result is in goodagreement with

therecentassociationof thec-lilsequencewith

one given stock of B77 ASV and was further

documented by analyzing other stocks of B77

ASV provided by different laboratories (listed

above). It was found that all viral RNAs that

were tested failed to hybridize with

cDNA,j,

(datanotshown).

ThecellularIU (c-il)sequence.v-lil couldbeof cellularorigin,since thehistoryof the B77 ASV

(lil) stockinvolvedrepeated passagesin CEFs.

To testthishypothesis, we hybridized cDNAti,

withDNAextractedfrom normal chickencells.

t32P]cDNArep

which hybridized with endoge-nous viral sequences was used as an internal standard. Inaparallel experiment,wemeasured

thereassociation kinetics ofchickenunique

se-quencesbyhybridizationof3H-labeled chicken

unique-sequence DNA with chicken DNA,

us-U

£6n

e

0

w __p

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.501.58.253.358.579.2]

lus. Recent data fromFrisbyet al. (4) show that

,-+

evsequences are notpresentinthe DNA from all

species

of the genus Gallus. Because we

,'1+

foundc-lilin the DNAfrom the domestic chick-en,weinvestigated other DNAsfrom the genus

Gallus. We

hybridized

cellular DNAs to

/

[3H]cDNA1il

under stringent conditions,

includ-ing

[32P]cDNArep

in all the kineticanalysesasa measureofhomology withev sequences (Table

' 3). As already shown(4), [32P]cDNArep

hybrid--/ ized to the DNA from a close ancestor of the

I' domestic chicken(redjungle fowl)but notto the

-

.,~'

DNA oftwoother

species

of the genus Gallus

,/'/(Sonnerat jungle/ fowland green jungle fowl). On

V ,' theother

hand,

cDNA1il

hybridized

totheDNA

,

,+_, from these two

species

tothesame extentas to

0-3

10-2

10-1 10o domestic chicken DNA. From this result, we

Crt(Mxs xl-1) concluded

_specific

that

c-lil

was a cellular sequence

for

species

ofthegenusGallus.

4. Hybridization of

cDNAil

with RNA from Relationshipofc-ilandv-il. Therelationship (isolated from the original tumor.

[3H]cDNA,i,

of the DNA sequences homologous to cDNA,1z

IcDNArep

were hybridized to dilutions of B77 in the chicken was analyzed by denaturing du-der stringent conditions. The percentage of plexes formed between cDNA11 and normal

wasmeasuredafter

Sl

digestion. Symbols:

chicken DNA. In addition, we denatured

du-A,11;

+,

[32PIcDNArep-

plexes between

3H-labeled

unique-sequence

chicken DNA and chicken DNA to obtain the

same internal standard. Both tritiated Tm value of chicken unique-sequence

dissocia-hybridized to cellular DNA with the tion. As described above, in separate

experi-netics (Fig. 5) (we standardizedwith the ments [32P]cDNArep was annealed to chicken

yve obtained after hybridization with DNA and thenwasaddedtothe DNA which had

cDNAe p; data not shown). This result

showedthatc-lilwaspresentin normalchicken

cellular DNA and behaved likeunique-sequence

DNA. The

Cotj12

value was 1,300 mol

sliter-1,

corresponding to about 1 copy of the c-lil

se-quence perhaploidgenome.

Hybridization of cDNAil to various avian

DNAs. Studieson the evolution ofc-srcand ev sequences have shown that these sequences

have evolved differently (8). We first analyzed

the distribution of c-lil in various avian species

by hybridization ofcDNA111 with cellular DNA

understringent conditions(0.6 MNaClat68°C,

Sl analysis) and at

Cot

values greater than 104

mol-s

liter-1

(10 times higher than the

C0t1/2

of

unique-sequence reassociation). cDNA,,1

hy-bridized to chicken DNA but not to the DNA

from differentrepresentatives of the family

Pha-sianidae (pheasant, quail, partridge, guinea

fowl, grouse) or from other families (turkey, emu), nor to DNA from XC cells (rat cells

transformedby RSV) or calf thymus (Table 2).

Tocompare the distribution ofc-liltothat of ev sequences, we hybridized

[32P]cDNArep

to the sameDNAs, since it was already shown (4) that ev sequences were extensively present only in

normal chicken DNA. In XCcells, we detected

the integratedprovirus with cDNArep

Comparative study of the origin of

RAV-0-relatedsequences andc-lilwithin the genus

Gal-c

_ U,

a)

50+

a)

a,

0

10' 103

Cot[Mxsxl'-1 1O4

FIG. 5. v-lil in normal chicken DNA. Denatured

normal chicken DNA (200 1Lg) was hybridized with

2,000 cpm of DNA per point ([3H]cDNA,i, or

3H-labeled unique-sequence chicken DNA). In both

ex-periments, [32P]cDNArep was added as an internal

standard (curves not represented). Hybridizations

wereconductedunder stringent conditionsat increas-ing Cot values to afinal Cot of 20,000 mols-liter-l.

Symbols: 0, [3H]cDNA,,,; A, 3H-labeled chicken

unique-sequenceDNA.

50

cc

.eCO)

cc

C,

FIG. 4 B77ASV and[32P] RNA un

annealing

[3H]cDN

ing the probes

samekii

Cqt

cur

930 BOCCARA ET AL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.501.59.231.60.267.2] [image:6.501.257.446.398.588.2]

[image:7.501.60.457.65.351.2]

c-lil, A CHICKEN CELLULAR SEQUENCE 931

TABLE 2. Hybridization of

cDNAjj,

to various normal avianDNAsa

Extent ofhybridization Approximate (%)with: phylogenetic

CellDNA

~~~~~~distance

Order Family Sub-family Genus

tested

from

[3HJcDNAj,,

[32P]cDNArep

domestic chicken(yr

X

106)

Galliformes Phasianidae Phasianini Gallus Domestic 58 48 0

chicken

Phasianus Ring-necked 3 7 30

pheasant

Chrysolophus Golden 3 2 30

pheasant

Lophura Silver 3 2 30

pheasant

Coturnicinae Coturnixex- Japanese 3 3 35-40

calfactoria quail

Perdicinae Perdix alec- Partridge 1 5 35-40 toris

Numidinae Numida Guinea fowl 1 1 35-40

Tetraoninae Lagopus Grouse 1 5 35-40

MeleagridinaeMeleagrininae Meleagris Turkey 1 1 40

Struthioniformae Dromiceius Emu 1 1 100

Control Rat-XC 1 54 300

(PrC pro-virus)

Calfthymus 1 1 300

aDNA wasextractedfrom liver (turkey, emu), from tissue culture cells (XC), from calf thymus, or from

10-

to 11-day-oldembryos (for all other considered species). Reaction mixtures containing denatured DNA (10 mg/ml),

[3HJcDNA,1j, and

[32P]cDNA,,p

were incubated at 68°C, and the percentage of annealing was assayed by S1

nucleasedigestion.The approximate phylogenetic distance from the domestic chicken was estimated by Prager etal. (10) on thebasis of the immunological relationship of transferrin and albumin.

been annealed to cDNAIil or to 3H-labeled

unique-sequence chicken DNA. Duplexes be-tween

cDNAjIt

and chicken DNA had a Tm of

88.5 ± PC, which is similartothe Tm value of

unique-sequence chickenDNA dissociation (89

± 1°C).

Expression of c-il innormal CEFs. Because

c-idhas been showntobeachickensequence,we

analyzed its level of expression in CEFs. Total

cellular RNA was hybridized to

[3H]cDNAIil,

andas a control to

[32P]cDNA,,p.

We found a

Crtlj2

of 500 mols Iiter-1 for the expression of

RAV-0-related sequences, which corresponds

to 80 copies per cell (the cells were chf+)

with a conventional value of

Crtj2

= 4 x l04

mol*s*liter-1 for1 copy percell. For c-lil

expres-TABLE 3. Distributionofc-lilamong DNAsfrom the genus Gallus'

Extentofhybridization (%)with: Phylogenetic distance

Genus Species Cell DNA from

[3H]cDNA,i

[(32PJcDNA,P

domestic chicken(yr

x 106)

Gallus Gallus Domestic chicken 58 48 0

(SPAFAS)

Redjungle fowl 54 45 0

Varius Greenjunglefowl 56 3 5

Sonnerati Sonneratjungle 57 1 0

fowl

Control Japanesequail 3 3 30

Calfthymus 1 1 300

a DNAwasextractedfromerythrocytes of exotic chickens, fromembryos(11 to15days old)of domestic chickens, orfrom calfthymus. Hybridizations wereperformed under stringentconditions atamaximumCot valueof20,000mol-s'liter-l.Theextentofannealingwasmeasured afterS1nucleasedigestion.Theapproximate phylogeneticdistance from the domestic chickenwasestimatedby Prageretal.(10).

VOL.43, 1982

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.501.60.458.510.640.2]

932 BOCCARA ET AL.

80H

70O

60-U)

Z50 =40

W-U)S

_

10_

80 85 90

Temperature(OC)

FIG. 6. Thermal denaturation profiles. Normal chicken DNA wasannealedto[3H]cDNA,,,orto 3H-labeledunique-sequencechickenDNA, and Tm values were determined asdescribed in the text. A duplex performed between [32P]cDNArepand normal chicken DNA was included in each Tm determination as an internal standard. Symbols: A,

[3H]cDNAj,,;

0, 3H-labeled chicken unique-sequence DNA; +,

[32P]cDNA,,p.

Numbers on the curves representthe experimental values ofTm.

sion, wefound a

Crt1/2

of2 x 104 mol-sliter-1

whichcorrespondsto1 to3copiesper cell(Fig.

7). Inaparallel experiment, usingtotal cellular

RNA from td B77 ALV-infected CEFs, we found the same Crt value forc-lilaswithnormal CEF cellularRNA, suggestingthat a retrovirus

10' 102 103 104

CrtMsx.I-l

FIG. 7. Transcriptionofc-lilinnormalCEFs.

To-talRNAwasextractedfromnormal CEFs and

hybrid-ized under stringent conditions to [3H]cDNA,i, and

[32P]cDNA,,p

toafinalC,t of 20,000mols-fliter' The extentofhybridizationwasmeasured by Si nuclease

digestion.Symbols: 0,[3H1cDNA,j,;+,[32P]cDNArep. ACrtl2 of4 x 104was arbitrarily taken for1 RNA

copyper averagecell.

I

B

[image:8.501.211.443.64.392.2]

Crt[M.sJ-'J

FIG. 8. Analysis of viral RNA and cellular DNA after infection ofquail cells by B77 ASV (lil).Japanese quail fibroblasts were infected at low multiplicity of infection by B77 ASV (lit). The cellswere passaged three timesand were fully infected and transformed before nucleic acid extraction. (A) High-molecular-weight DNA was prepared as describedpreviously(3). The DNA was sonicated tofragments approximately 500nucleotideslong,asestimatedbyelectrophoresis in1% agarose gel withaHindllldigestionofpolyoma DNAas amarker. The DNA wasfully denaturedby boilingfor 5 min before annealingwith cDNA. The DNA (finalconcentration, 10 mg/ml) was hybridized to

[3H]cDNAj,,

(0) and

[32P]cDNA,,p

(+) toreach a maximumCotvalue of 2 x 104 mol sliter-1at68°C in 0.6 M NaCl at various times in glass capillaries (Brandt). The extent of annealing was measured by S1 nucleasedigestion. (B) Thesupernatant was collected, andviral RNA was extracted as described previously (19, 21). The RNA pellet dissolved in 0.6 M NaCl buffer was further diluted and hybridized to [3H]cDNA,1,(0) and

[32P]cDNA,rp

(+) at 68°Cfor 24 h in7,ul of the NaCl buffer. The extent of hybridization was measured byresistance of the cDNA to hydrolysis byS1 nuclease.

lacking thev-lil sequencesisnot able toinduce

the cellular c-lil sequences in infected CEFs

(data not shown).

v-il is a transmissible sequence. Our

on November 10, 2019 by guest

http://jvi.asm.org/

[image:8.501.57.248.467.602.2]

c-lil, A CHICKEN CELLULAR SEQUENCE 933

mentsfavoredthehypothesis that the B77 ASV

(lit) contained a transmissible new sequence but could not exclude the specific induction of the c-fil sequences at each infection cycle, which was

followed by the packaging of such transcripts.

We thus infected with the B77 ASV (lit) quail

fibroblasts that did not contain the c-lil

se-quences in their genome (see Table 2). The

results are shown in Fig. 8. Indeed, the v-lil

sequences were retrotranscribed, found (Fig.

8A) in the DNA (about 1 copy per haploid

genome), and transcribed as efficiently as the

other viral sequences (Fig. 8B). Thus, the

tro-pismof thev-lil sequences extends to host cells

that do not contain the homologous cellular sequences in their genomes.

DISCUSSION

Inthis paper, we have shown that a

chicken-specificcellular sequence

called

c-lilhasbecome

associatedwith a given stock of B77 ASV. From

our stock of B77 ASV which had been

main-tained in vitro, we prepared a cDNA111 by a previously described selection procedure (19).

Its genetic complexity was measured by the

Cot11

ofhybridization between an excess of 3H-labeledcDNA111andits homologous

nonradioac-tiveRNA;the

cDNA1j1

wasestimated to contain

2,250 nucleotides ± 10%.

The v-lil sequence was shown to be present

onlyin our stockof B77 ASV, not in other avian

retroviruses, irrespective of their group or

sub-group: we did not find v-lil associated with

RSVs, ALVs, orDLVs, regardless of whether

they were from subgroup B, C, D, or E. We havetriedtoelucidatethe naturalhistoryof this

stockof virus. v-lilwas notassociated with the

B77 ASVisolated fromtheoriginaltumor.

Like-wise, various B77 ASV stocks from different

laboratories did not containv-lil. Wethus

con-clude that this sequence has been

acquired

re-cently by cell culture

passaging

ofa

particular

stock ofB77 ASV. This

specific

acquisition

is

probably a rare eventsinceitwas notobserved

in any of the other viral strains

tested,

but

recombinations ofother sequences with

retro-viralgenomesmighthaveoccurredin other viral

stocks.

Because our stock was passaged in chicken

cells, we determined whether

cDNA,j,

hybrid-ized with a chicken cellular sequence. We

showed thatourprobe

hybridized

toDNAfrom

uninfecteddomesticchickens. This sequenceis

absent from the DNA of various membersof the

family Phasianidae

(pheasant,

quail,

partridge,

guineafowl, and

grouse)

and from the DNAof

other families

(turkey, emu)

as

well

as from

mammalianDNAs(rat

XC,

calf

thymus);

there-fore, c-lil is not a

highly

conserved sequence.

This resultdifferentiates c-lil from c-src and all

other avianc-oncsequences sofar characterized

(12, 15-17).

To define the origin ofc-lil in chickens, we

hybridized cDNA,l to the DNA from various exotic chickens belonging to the genus Gallus.

Unlike ev

sequences,

which are found only in

somespecies (4), c-lil is present in the DNA of

all tested chickens from the genus Gallus, and

thus appears to be characteristic of this genus.

The v-lil associated with B77 ASV has

changed little from its cellular counterpart,since

theTmvalue for duplexes between

cDNAIil

and

chicken DNA was similar to the Tm value of

chickenunique-sequencedissociation. This

sug-gests that the percentage of mutation of v-lil

during the course ofpropagation of B77 ASV

(lit) must have been low.

What mechanism can we propose for the

appearance of c-lil during the in vitro passageof

B77 ASV (clone 9)? This might have occurred

through packaging of a major

sp;ries

of RNA

present in tissue culture cells as virus-like 30S

RNAin mouse cells (2). However, c-lil is

tran-scribed at a very low level in normal chicken

fibroblasts (1 to 3 copies per cell), and there is no increase of c-lil transcription in CEFs infected

with td B77 ALV. Moreover,c-lilacquisition by

simple packaging seems unlikely, as the B77

ASV(lit) stock has been passaged many times at

low multiplicity of infection on CEFs or even in

quailfibroblasts (that do not contain the

homolo-gousc-lil sequences in their DNA) and has still

conserved v-lil. It is also unlikely that v-lil

sequences represent an autonomously

replicat-ing virus, since their complexity of ca. 2

kilo-basesis quite small for a complete viral genome.

This suggests that v-lil is transmissible because

it is covalently linked to viral RNA from B77

ASV or its td B77 accompanying mutant.

In-deed, preliminary evidence suggests that the lil

sequences arecovalently inserted next to the src

gene in the recombinant virus (manuscript in

preparation). So far, we have no clues to the

function, ifany, conferred to our virus bythe

presence of this additional piece of nucleotide

sequences. This will require cloning these

se-quences deprived of the src transforming

se-quences and studying the biological properties

of thisvirus.

ACKNOWLEDGMENTS

We thank theresearchersalreadymentioned in the text for

providing biological materials to us. We also thank Simon Saule and Edward Stavnezer for helpful discussions, and ChristianLagroufor excellent technical assistance.

This workwassupported by grantsfromInstitutNational de la Sant6etde la RechercheM6dicale(CRL 802036, CRL

812043), Centre National de la Recherche Scientifique (AI 033695),D61egation Generaleala Recherche Scientifique et

Technique(81L0725),and the Pasteur Institute of Lille.

VOL. 1982

on November 10, 2019 by guest

http://jvi.asm.org/

934 BOCCARA ET AL.

LITERATURECITED

1. Astrln, S. M., E. G. Buss, and W. S. Hayward. 1979. Endogenous viralgenesarenon-essentialin thechicken. Nature(London)232:339-341.

2. Besner,P.,U.Obbevsky,D.Baltimore,D.Dolberg, and H. Fan.1979. Virus-like30S RNA inmousecells. J.Virol. 29:1168-1176.

3. Bishop,J. M.1978. Retroviruses. Annu. Rev. Biochem. 47:35-88.

4. Frisby, D. P., R. A.Weiss, M. Roussel,and D.Stehelin. 1979. Thedistribution of endogenous chicken retrovirus

sequencesin theDNA of Galliform birds doesnot coin-cidewith avianphylogenetic relationships. Cell 17:623-634.

5. Graf, T., and H. Beug. 1978. Avian leukemia viruses.

Interaction with theirtarget cells in vivo and in vitro. Biochim.Biophys. Acta 516:269-299.

6. Hanafusa, H. 1977. Cell transformationby RNA tumor

viruses, p. 401-458. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Comprehensive virology: regulation and

genetic viral gene expression and integration, vol. 10. PlenumPublishing Corp., New York.

7. Ha1f1s, T., H. Hanas, and T. Mlyamoto. 1970.

Recoveryofanewvirus fromapparently normal chick cells by infection with avian tumorviruses. Proc. NatI. Acad.Sci.U.S.A. 67:1797-1803.

8. Hughes, S. H., F.Payvar,D.Spector, R.T.Schlmke,H. L.

Robson, G. S.Payne,J.M.Bishop, and H. E. Varmus. 1979. Heterogeneityofgenetic loci in chickens:analysis ofendogenous viral and nonviralgenes by cleavage of DNAwith restriction endonucleases. Cell 18:347-359. 9. Leong,J.-A.,A.-C.Garapln, N.Jaclkon,L.Fanshler, W.

Levnson,andJ.M.Bisbop. 1972.Virus-specific ribonu-cleic acid in cells producing Roussarcomavirus: detec-tion and characterizadetec-tion.J.Virol.9:891-902.

10. Prager, E. M., A. H. Brush, R.A.Nolan,M.Nakanishi,

andA.C. Wilson. 1974. Slow evolution of transferrinand

albumin in birdsaccordingtomicrocomplementfixation

analysis. J. Mol. Evol. 3:243-262.

11. Robinson, H. L. 1978. Inheritance and expression of chickengenesthatarerelatedtoavian leukosissarcoma

virusgenes.Curr. Top. Microbiol. Immunol.83:1-37.

12. Rousel, M., S. Saule, C. IAgrou, C.Rommuns, H. Beug, T.Graf, and D. Stebheln. 1979. Threenewtypesof viral

oncogeneof cellularorigin specific forhaematopoietic cell transformation. Nature(London)281:452-459. 13. Saule, S., M. Rosel, C.Lagrou,and D.Stehelin.1981.

Characterization of theoncogene(erb) of avian erythro-blastosis virus and its cellular progenitor. J. Virol. 38:409-419.

14. Shank, P. R., S. H. Hughes, H.J.Kung,J.E.Majors, N.

Quintrell, R. V. Guntaka, J. M. Bishop, and H. E.

J.VIROL. Varmus.1978.Mapping unintegratedaviansarcomavirus DNA:termini of linear DNA bear 300nucleotidespresent once or twice in two species of circular DNA. Cell

15:1383-1395.

15. Sheiness, D.,andJ.M.BIsbop.1979.DNA and RNA from uninfected vertebrate cells contain nucleotide sequences relatedtotheputativetransforminggeneof avian myelo-cytomatosisvirus.J. Virol.31:514-521.

16. Shlbuya,M.,T. Hanafusa, H.Hanafusa, and J. R.

Ste-phenson.1980.Homologyexists among thetransforming

sequences ofavian and feline sarcoma viruses. Proc. Natl.Acad. Sci. U.S.A.77:6536-6540.

17. Spector,D. H.,H. E.Varmus,andJ.M.Bishop. 1978.

Nucleotidesequencesrelated tothetransforminggeneof avian sarcomavirus arepresent in DNA of uninfected vertebrates. Proc. Natl. Acad. Sci. U.S.A. 75:4102-4106. 18. Stavuezer,E., D. S. Gerhard, R. C. Binari, and I.Balm.

1981. Generationoftransformingviruses in cultures of chicken fibroblasts infected with an avianleukosisvirus. J.Virol.39:920-934.

19. SteheUn, D.,R. V. Guntaka,H. E. Varmus, and J. M.

Bishop. 1976. Purification of DNA complementary to

nucleotide sequences required for neoplast

transforma-tion offibroblastsbyavian sarcomaviruses.J.Mol. Biol.

101:349-365.

20. StebeUn, D.,H. E.Varmus, J.M.Bisbhop,andP.K.Vogt.

1976. DNA relatedtothetransforming gene(s)ofavian

sarcomaviruses is present in normal avian DNA. Nature

(London)260:170-178.

21. Taylor, J. M., R. ilMmensee, and J. Summers. 1976. Efficient transcription of RNA into DNA by avian

sarco-maviruspolymerase. Biochim. Biophys. Acta 442:324-330.

22. Vlgne,R.,M. L.Breltman, C. Moscovici, and P. K.Vogt.

1979. Restitutionoffibroblast-transforming ability insrc

deletion mutants of avian sarcoma virus during animal passage.Virology93:413-426.

23. Vogt,P.K., and R. R. Frils. 1971. An avian leukosis virus related to RSV(0): properties and evidence for helper

activity. Virology 43:223-234.

24. Wang, L. H., C. C. Halpern, M. Nadel, and H. Hanafusa. 1978. Recombination between viral and cellular se-quences generates transforming sarcoma virus. Proc. NatI. Acad. Sci. U.S.A. 75:5812-5816.

25. Weiss,R.A., W. S. Mason, and P. K. Vogt. 1973.Genetic

recombinants and heterozygotes derived from endoge-nousandexogenousavian RNA tumor viruses. Virology S2:535-552.

26. Young,B. D., P. R. Harrison, R. S.Gilmour,G. D.Blrnie, A. Hell, S.Humphries,and J. Paul. 1974. Kinetic studies ofgenefrequency.II.Complexityofglobin complemen-taryDNA and its hybridization characteristics.J. Mol. Biol.84:555-568.

on November 10, 2019 by guest

http://jvi.asm.org/